Oxidative Stress Resistance Mechanisms in Filifactor Alocis

Filifactor Alocis 的氧化应激抵抗机制

• 批准号: 10217430
• 负责人: Arunima Mishra
• 金额: $ 15.8万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217430
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Adult; Alleles; Anaerobic Bacteria; Bacteria; Basic Science; Biological Models; Cell physiology; Cells; Coculture Techniques; Communities; Complex; Data; Development; Diagnostic; Disease; Endodontics; Environment; Etiology; Exposure to; Forsythia; Fusobacterium; Gene Expression Profile; Genes; Genetic; Genome; Goals; Growth; Health; Homeostasis; Human; Hydrogen Peroxide; Infection; Inflammatory; Light; Manganese; Molecular; Molecular Genetics; Mutagenesis; Oral; Organism; Oxidative Stress; Pathogenesis; Pathogenicity; Patients; Periodontal Diseases; Periodontal Pocket; Periodontitis; Phylogenetic Analysis; Play; Porphyromonas gingivalis; Prevention; Prophylactic treatment; Reactive Oxygen Species; Refractory; Reporting; Research; Resistance; Ribosomal RNA; Rod; Role; Severity of illness; Stress; Structure of gingival sulcus; System; Treponema; Treponema denticola; Virulence; Virulence Factors; bacterial community; base; diagnostic biomarker; emerging pathogen; genetic manipulation; innovation; microbial community; microbiome research; new therapeutic target; novel therapeutic intervention; novel therapeutics; oral microbiome; pathogen; periodontopathogen; resistance mechanism; response; tool; transcriptome sequencing; virtual

A. PROJECT SUMMARY/ABSTRACT Recent oral microbiome studies have recognized a myriad of as-yet-culturable and fastidious organisms that have shown a strong correlation with periodontal disease severity. It is likely that the emerging new pathogens may play a more significant role in the disease compared to the traditional “red complex” bacteria Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. One such previously unrecognized organism, Filifactor alocis, is a Gram-positive, asaccharolytic, obligate anaerobic rod. Several recent studies have found this bacterium at significantly higher levels in adult or refractory periodontitis patients and have suggested that it could be included as a diagnostic indicator of periodontal disease. Currently, there is little or no information on survival mechanisms and virulence of F. alocis. Primarily, this is due to the unavailability of an efficient genetic system to allow genetic manipulations of the F. alocis genome. In response to environmental stress, our preliminary studies showed that F. alocis is relatively more resistant to H2O2-induced oxidative stress compared to P. gingivalis. Also, under H2O2-induced stress conditions, the survival of P. gingivalis is enhanced more than 4-fold in the presence of F. alocis. These observations suggest that F. alocis may have the ability to modify/reduce the oxidative stress environment and stabilize the microbial community of the periodontal pocket. In an RNA-seq analysis, the transcriptional profile of F. alocis showed that in coculture with P. gingivalis (compared to F. alocis monoculture) under H2O2-induced stress, the most highly upregulated genes in F. alocis encode for a putative manganese ABC transporter FA0894-FA0895-FA0896-FA0897. Manganese has been proposed to detoxify reactive oxygen species and protect bacteria from oxidative stress. It is our hypothesis that the F. alocis hypothetical ATP transporter, FA0894-FA0897, may play an important role in enhanced protection/survival of P. gingivalis against H2O2-induced stress. We wish to understand the modulation of F. alocis potential virulence factors in response to interaction with P. gingivalis and evaluate if they contribute to protection/persistence of P. gingivalis against the oxidative environment of the periodontal community. The proposed specific aims are: (1) to evaluate the specific role(s) of F. alocis putative ABC transporter FA0894-FA0897 in the protection/survival of P. gingivalis under H2O2-induced stress. (2) to develop an efficient genetic system for Filifactor that includes a markerless, in-frame deletion system and/or transposon mutagenesis system. Collectively, the data generated will facilitate a comprehensive assessment of the molecular mechanism(s) and overall interplay involving F. alocis and the ‘keystone’ pathogen P. gingivalis. It will also generate a polymicrobial model system that may facilitate the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease. Furthermore, the new genetic manipulation system will help to uncover the relative significance of F. alocis in the etiology of periodontal disease.

A. 项目概要/摘要 最近的口腔微生物组研究已经认识到许多尚未培养且挑剔的微生物 已显示与牙周病严重程度密切相关的微生物很可能正在出现。 与传统的“红色复合体”相比，新的病原体可能在疾病中发挥更重要的作用 牙龈卟啉单胞菌、连翘坦纳菌和齿垢密螺旋体就是其中之一。 未被识别的生物体 Filifactor alocis 是一种革兰氏阳性、解糖、专性厌氧杆菌。 最近的研究发现，这种细菌在成人或难治性牙周炎患者中的水平显着较高 并建议将其作为牙周病的诊断指标。 目前，关于 F. alocis 的生存机制和毒力的信息很少或根本没有。 这主要是由于缺乏有效的遗传系统来对F. alocis基因组在应对环境胁迫方面，我们的初步研究表明F. alocis相对较弱。 与 H2O2 诱导的应激相比，牙龈卟啉单胞菌对 H2O2 诱导的氧化应激具有更强的抵抗力。 在 F. alocis 存在的条件下，牙龈卟啉单胞菌的存活率提高了 4 倍以上。 观察结果表明，F. alocis 可能具有改变/减少氧化应激环境的能力，并且 在RNA-seq分析中，牙周袋的微生物群落的转录谱。 F. alocis 的结果表明，在 H2O2 诱导下与 P. gingivalis 共培养（与 F. alocis 单一培养相比） F. alocis 中上调最高的基因编码假定的锰 ABC 转运蛋白 FA0894-FA0895-FA0896-FA0897 已被提议可解毒活性氧和 我们的假设是 F. alocis 假设的 ATP 转运蛋白， FA0894-FA0897，可能在增强牙龈卟啉单胞菌的保护/存活方面发挥重要作用 我们希望了解 F. alocis 潜在毒力因子的调节。 对与牙龈卟啉单胞菌相互作用的反应并评估它们是否有助于保护/持久性牙龈卟啉单胞菌。 gingivalis对抗牙周群的氧化环境提出的具体目标是：（1） 评估 F. alocis 假定的 ABC 转运蛋白 FA0894-FA0897 在保护/生存中的具体作用 P. gingivalis 在 H2O2 诱导的胁迫下 (2) 开发一个有效的 Filifactor 遗传系统，其中包括 无标记的框内删除系统和/或转座子诱变系统。 总的来说，生成的数据将有助于对分子的全面评估 涉及 F. alocis 和“关键”病原体 P. gingivalis 的机制和总体相互作用。 生成可能促进新型治疗干预措施开发的多微生物模型系统 此外，新的基因操纵系统有助于控制和预防牙周病。 将有助于揭示 F. alocis 在牙周病病因学中的相对重要性。